Vertebrate tissue matrices contain hyaluronan (HA), which modulates cell behavior (e.g. during development, cancer, wound healing and angiogenesis). Humans turnover grams of HA daily, and accumulating data suggest that alterations in the normal HA-turnover process can be critical in disease pathogenesis. Also, HA that is smaller than normal has unexpected biological activities in inflammatory responses and cancer-cell sensitivity to chemotherapeutics. It is not yet known if small HA arises in vivo by degradation or synthesis. The long-term hypothesis guiding this project is that HA synthesis must be tightly controlled in order to achieve normal homeostasis and health. HA is synthesized by HA synthase (HAS), first cloned under this project by the principal investigator. Many diseases, including birth defects, arthritis, ulcerating wounds, and some cancers may arise from, or be promoted by, altered HAS function. Our goal is to under- stand the molecular details of how the 3 human HAS isozymes make HA, regulate HA size, and how HA is transferred through membranes to the matrix or to the cell surface as an HA "coat". A major obstacle to progress is that detergents inactive the human isozymes, and native, active human HAS cannot yet be specifically immuno-purified or localized in cells. We will use recent findings about the lipid-dependence of streptococcal HAS to establish conditions to detergent-solubilize human HAS1, 2 and 3 with retention of activity. Our strategy is to use structure-function analyses of streptococcal HAS to elucidate common mechanisms within the Class I HAS family, in order to understand how the human HASs function and how to purify active human HAS. A major project focus during this renewal period is to determine if bacterial and human HASs export HA from the cytosol to the cell surface or ECM by a Pore or ABC-transporter mechanism, or by both. Two Pore Hypotheses to be tested are that HAS itself creates an intra-protein pore, and that HAS translocates HA vectorally through its pore during biosynthesis. We will also test three molecular predictions of the ABC- transporter Hypothesis for HA export. The following specific aims address multiple hypotheses. 1) To characterize the pore activity of HAS. 2) To determine if HAS translocates HA vectorally across membranes. 3) To determine key features of the mechanism of HA synthesis by HAS. 4) To test three molecular predictions of the hypothesis that ABC-transporters export HA. 5) To overcome two obstacles that hinder purification of active human HASs. Public Health Relevance: Many diseases, including birth defects, arthritis, ulcerating wounds, and some cancers may arise from, or be promoted by, altered synthesis of hyaluronan (HA) in the body. HA is synthesized by HA synthases, which were first cloned under this project by the principal investigator. Knowing details about how the three human HA synthases function will enable us and others to develop strategies to identify modulators and inhibitors of these enzymes (e.g. to alter HA activity, HA size or cellular localization) that might be drug candidates for clinical applications, particularly in cancer, metastasis, and streptococcal diseases.